Embryonic stem (ES) cells are stem cells established from early embryos which may be cultured over a long period of time while maintaining pluripotentiality; i.e. the ability to differentiate into any cell type of the body. Using these properties, human embryonic stem cells may be used for cell transplantation therapies for various diseases such as Parkinson's disease, juvenile diabetes, leukemia, and other debilitating human diseases. However, transplantation of ES cells may result in rejection in the same manner as organ transplantation. Moreover, from an ethical viewpoint, there are many who contest the use of ES cells, which are established by destroying human embryos.
If dedifferentiation of patients' own differentiated somatic cells could be induced to establish cells having pluripotency and growth ability similar to those of ES cells (hereinafter, “induced pluripotent stem cells” or “iPS cells,”), they would not only offer the prospect of producing patient-specific cells but also circumvent the ethical conundrum that surrounds the isolation of human embryonic stem (ES) cells from early embryos.
Induced pluripotent stem (iPS) cells have been generated using Oct3/4, Sox2, Klf4, and c-Myc (SOKM; Takahashi et al., Cell 126:663-76, 2006). However, published methods for nuclear reprogramming mediated by the introduction of genes are challenged by low efficiency, in which only a small number of induced pluripotent stem cells can be obtained, and by a slow time course (>7 days). In addition, some of the factors are oncogenic (c-Myc and others). Therefore, a method of generating iPS cells from somatic cells for gene therapy and other uses at high efficiency while avoiding the use of tumorogenic factors is lacking in the art.
MicroRNAs (miRNAs) are small endogenous RNA molecules (˜21-25 nt) that to regulate gene expression by targeting one or more mRNAs for translational repression or cleavage. They are small inhibitory RNAs capable of suppressing the translation of target genes with high complementarity. Several thousand miRNAs have been identified in organisms as diverse as viruses, worms, and primates through cloning or computational prediction.
The miR-302-367 cluster has high intracellular abundance and is cell type specific to embryonic stem cells. This miRNA-302-367 cluster was initially identified from cDNA libraries generated by directional cloning using size-fractionated RNA (17-26 nt) from undifferentiated hESCs. This cluster is codified in the human chromosome 4 and comprises nine different miRNAs co-transcribed in a polycistronic manner: miR-302a, miR-302a*, miR-302b, miR-302b*, miR-302c, miR-302c*, miR-302d, miR-367 and miR-367*. The miR-302 family contains seven miRNAs with a highly conserved 5′ region. The miR-302-367 cluster was first identified to be expressed in mESC, hESC and in their malignant counterparts hECCs.
The miR-302-367 genes can target over 445 human genes, most of which are developmental signals involving the initiation and/or facilitation of lineage-specific cell differentiation during early human embryogenesis. These target genes are listed in the target prediction sites linked to the miRBase::Sequences program at the Sanger website, including TARGETSCAN and PICTAR-VERT.
It is unknown if the miR-302-367 cluster can reprogram normal somatic cells at high efficiency. There exists a need for improved miRNA-based compositions and methods for inducing pluripotent cells.